1. Field of the Invention
The present invention relates to a method of introducing tritium into various compounds. More particularly, the present invention relates to a method of introducing tritium into sensitive biological molecules such as proteins, peptides, nucleic acids, polysaccharides, amino acids, polymers, inorganic compounds as well as simple organic molecules.
2. Description of the Prior Art
Tritium labeled compounds are widely used in medicine and in the study of biological problems. The advantage of using tritium as a label resides in the low cost of the isotope and the high specific radioactivity that can be achieved in the labeled material. Because of the ubiquitous nature of hydrogen in organic compounds, almost any compound of medical or biological interest can potentially be labeled with tritium. Substitution of tritium atoms for hydrogen usually results in no significant alteration in the chemical or biological properties of the compound labeled.
The incorporation of tritium into organic molecules including proteins can be achieved by synthetic, biosynthetic and non-synthetic methods. Non-synthetic methods include: (1) self-irradiation of a substance with tritium (Wilzbach labeling), (2) excitation and ionization of a substance induced by microwaves, an electrical discharge, .gamma.-ray irradiation, or the like (modifications of the Wilzbach procedure), (3) recoil tritiums from nuclear reactions, (4) free radical interceptor methods, and (5) exchange reactions catalyzed by acid, bases or other catalysts. Most of these methods have been developed for use with simple organic compounds. However, very few of the methods have been applied to the labeling of complex biological molecules such as proteins, nucleic acids, lipids or polysaccharides. In addition simpler organic molecules which are difficult to isolate or synthesize can only rarely be obtained in a tritiated form using these methods.
Most tritium labeled proteins, nucleic acids, lipids and polysaccharides are produced by biosynthetic methods or by chemical modification. Tritium can be incorporated into biological molecules by these methods often with very high specific activities. However, these methods do not find general utility and are limited to particular systems. The development of a biosynthetic method for the production of a labeled protein is usually more difficult than the isolation of the protein itself and is quite time consuming. In fact, human proteins are difficult or impossible to obtain labeled by biosynthetic methods. Chemical methods for incorporation of labels into such proteins often yield impure products or result in alteration or loss of biological activity.
Non-synthetic methods of tritium labeling have been applied to proteins, but with only limited success. This is evident from the fact that there is only a small number of recent literature citations in the major scientific journals describing non-synthetic tritium labeling methods. In addition, an examination of catalogs of major suppliers of radiochemicals reveals that almost no tritium labeled proteins are commercially available. In fact, the only proteins that are available in any significant number are those which have been labeled with iodine-125. The available nucleic acids, lipids and polysaccharides have usually been produced biosynthetically by bacteria.
Wilzbach (J. Am. Chem. Soc. 78, 5132 (1956) and 79, 1013 (1957) originally reported that organic compounds can incorporate tritium if exposed to large amounts of tritium gas for long periods of time. In general, the incorporation of tritium into sugars, steroids and aromatic hydrocarbons is relatively good, whereas in the case of polypeptides and aliphatic hydrocarbons, the tritium incorporation is relatively poor. The labeling technique of Wilzbach results in considerable destruction of the sample and in the case of amino acids, it has been shown to result in partial recemization and reduction of the aromatic ring of phenylalinine (Parmentier, J. Label, Compd. 1, 93 (1965) and 2, 367 (1966)).
The proteins lysozyme and ribonuclease have been labeled by the Wilzbach procedure (Steinberg et al., Science 126, 448 (1957)). With lysozyme, only half of the nonexchangeable tritium was found in the active enzyme peak upon chromatography, showing that extensive decomposition had occurred in the labeling reaction. Likewise, ribonuclease was obtained with 82% enzyme activity and again was separated from a degraded fraction of high specific radioactivity.
The peptide hormones insulin and oxytocin have both been labeled by the Wilzbach procedure. With insulin, repeated crystallization or electrophoresis and crystallization was necessary to purify the protein (Von Holt et al., Biochem, biophys. Acta 38, 88 (1960)). Oxytocin labeled by the Wilzbach method had a specific activity of 12 .mu.Ci/mg (Du Vigneaud et al., J. Am. Chem. Soc. 84, 409 (1962)). After electrophoresis only a small fraction of the radioactivity was bound by the hormone. All of the component amino acids were labeled. The biological potency of the purified oxytocin was only 60% of nonlabeled oxytocin.
There have been many other attempts to apply the Wilzbach procedure to proteins, and it appears that the successful attempts have been substantially outnumbered by failures. In many cases the protein is inactive after labeling (gonadotropin, .gamma.-globulin, or tetanus toxin) and in these cases no purification was attempted or no biological activities were measured. Thus, it seems that the Wilzbach procedure results in gross destruction of the protein with the generation of multiple impurities. The few proteins which were carefully purified showed low specific activities. Thus, this method has very limited usefulness.
A number of procedures have been developed which are modifications of the procedure by Wilzbach. These procedures involve exposure of the compound to tritium gas followed by activation of the tritium with either an electrical discharge or by a microwave generator.
Fisher et al, U.S. Pat. No. 3,238,139, discloses a process for incorporating tritium into organic compounds by subjecting organic compounds to an electric discharge in the presence of tritium gas. Fisher et al suggests that tritium incorporation occurs primarily through reaction of organic radicals formed by the electric discharge and tritium gas.
Labeling techniques using an electrical discharge method were first applied to simple organic molecules (Dorfman and Wilzbach, J. Phys. Chem. 63, 799 (1959)), but these techniques appear to present formidable problems with proteins. Angiotensin II, a small peptide, has been labeled using an electric arc and after purification (40% recovery) exhibited undiminished pressor and oxytoxic activity (Khairallah et al., Arch. Biochem. Biophys. 171, 729 (1962)). Recently, Noyer et al., J. Label. Compd. Radiopharm. 72, 365 (1976) have reported the labeling of ribonuclease in such a manner. Even though high specific activities (2-10 Ci/mmol) were achieved, extensive decomposition and low enzyme activities (32-68%) were also obtained. Moreover, it is not clear whether the radioactivity is associated with the active enzyme or the inactive decomposition products. Still further, Wolfgang et al., J.A.C.S. 78, 5132 (1956) disclose a method of labeling in which a substance in a tritium atmosphere is subjected to an electric discharge between electrodes of a potential difference of 500 volts.
A range of organic compounds, including amino acids, peptides and proteins, has been tritiated utilizing microwave discharge activation of tritium gas as disclosed by Hembree et al., J. Biol. Chem. 239, 3741 (1973). A free radical mechanism is probably responsible for tritium incorporation in this discharge method. High specific activities (up to 15 Ci/mmol) were obtained for many of the compounds tritiated by the microwave discharge method, but extensive purification of the products obtained was required. For each compound studied, the crude preparation after the removal of easily exchangeable tritium contained impurities of high specific activity which represented from 50 to 90% of the residual tritium.
Three peptides (H-Leu-Tyr-Leu-OH, H-Ile-Ile-D-Val-OH and H-Val-Ala-Ala-Phe-OH) were tritiated by this method as disclosed by Hembree et al., J. Biol. Chem. 248, 5532 (1973) to specific activities of 0.1-2.0 Ci/mmole after purification. The protein hormones adrenocorticotropin (ACTH), luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were also tritiated. The ACTH preparations had specific activities of 0.3-14.5 Ci/mmole. The specific activities of the products were highest when small samples were tritiated and were 1000 times greater than the activities obtained from the technique employed by Wilzbach using similar amounts of ACTH.
It is apparent from the above discussion that the microwave discharge procedure represents a significant advance in the field of tritium labeling of proteins and other biological molecules. The highest incorporation of tritium in a molecule (0.5 tritium atoms per molecule) has been achieved by this procedure. Yet, extensive decomposition of the substrate molecules still occurs during labeling under microwave discharge conditions. This is not unexpected since it is well known that the action of ionizing radiation on proteins causes main chain disruptions, polymerizations, chemical modifications of side chains and results in severe conformational damage. Separation of highly radioactive by-products from the labeled protein limits the usefulness of this procedure.
In the free radical interceptor method the protein is first irradiated with .gamma.-rays or exposed to an electrical discharge from a Tesla coil. Free radicals are formed and subsequent exposure to tritiated hydrogen sulfide yields a tritiated protein. Exchangeable tritium is then removed by dialysis. Lysozyme and ribonuclease have both been tritiated by this procedure using .gamma.-irradiation as shown by White and Riecz, Biochem. Biophys. Res. Comm. 30, 303 (1968). In each case, chromatography of the reaction product revealed the presence of impurities formed in the reaction; however, the major peak is reported to have the full enzyme activity. Similar results were obtained with lysozyme, ribonuclease and actin which were tritiated using an electrical discharge as shown by White et al., Anal. Biochem. 30, 245 (1969). Repeated crystallization of the lysozyme resulted in a drop in the specific activity from 40 to 13 Ci/mole. Since the enzyme activity did not change, it was thought that the procedure was simply removing some exchangeable tritium. In contrast, the enzyme activity of labeled lysozyme prepared by the technique of Wilzbach was substantially altered by crystallization.
The problems of the previously discussed tritiation methods are shared by the free radical interceptor method. Free radicals are extremely reactive and can undergo many types of reactions, most of which would degrade the protein. In addition, the method does not appear to be very reproducable.
Still other attempts have been made to incorporate tritium into a variety of protein substrates by several of the techniques discussed above as summarized in Table 1 below.
TABLE 1 __________________________________________________________________________ Tritium Labeling of Proteins by Gas Exposure Methods Sp. Act. mole tritium/ Compound Tritiation Method .mu.Ci/mg mole substrate References __________________________________________________________________________ ACTH Wilzbach 1.8 0.00025 Nishizawa et al., Can. J. Biochem. 43, 1489 (1965) Lysozyme Wilzbach -- 0.0018 Steinberg et al., Science 126, 447 ribonuclease (1957). Insulin Wilzbach 4.6 0.00083 Von Holt et al., Biochim. biophys. Acta 38, 88 (1960) Von Holt and Von Holt, Naturwissen- schaften 45, 289 (1958) Thyrotropin releasing Wilzbach 8000 0.1 Schally and Redding, Int. J. Appl. hormone (TRH) Radiat. Isotopes 21, 742 (1970) (a tripeptide) Vasopressin Electrical discharge 400 0.01 Fong et al., Proc. Nat. Acad. Sci. 46 1273 (1960) Angiotensin II Electric arc 300 -- Khairallah et al., Science 138, 523 (1962) Ribonuclease Electrical discharge -- 0.09-0.3 M. Noyer et al., J. Labeled Cmpd. Radiopharm. 12, 365 (1976) Adenocorticotropin Microwave discharge 3200 0.5 Hambree et al., J. Biol. Chem. 248, (ACTH) 5532 (1973) .beta..sub.2 -microglobulin Microwave discharge 500-5000 -- Wassels et al., Radiat. Res. 74, 35 (1978) .beta.-lipoprotein Tritium exchange with 0.72 -- Cosztonyi et al., Nature 208, 381 CH.sub.3 CO.sub.2 T (1965) Lysozyme Free radical 0.7 0.00033 White and Riecz, Biochem. Biophys. ribonuclease interruptor (.gamma.,HST) Res. Comm. 30, 303 (1968) Lysozyme Free Radical 0.4-0.06 .003 White et al., Anal. Bioch. 30, 295 ribonuclease interceptor (.gamma.,electrical (1969) actin discharge, HST) Collagen Free radical 0.7 0.0074 Labrosse et al., Anal. Bioch. 70, 218 interruptor (electrical (1976) discharge, HST) __________________________________________________________________________
In view of the disadvantages of the above-described tritiation techniques, a need continues to exist for a method of introducing tritium into organic compounds, especially sensitive biological molecules such as proteins, peptides, lipids, nucleic acids and polysaccharides as well as simple organic compounds which are particularly difficult to isolate or synthesize, which does not result in structural modification, loss of activity and deterioration of the substrate molecules.